We propose to continue to develop and capitalize upon the ability of T4 RNA ligase to serve as a reagent for nucleic acid synthesis and modification. The enzyme is capable of joining single strand deoxyoligoribonucleotides and of adding a single 2' -deoxyribonucleoside -3', 5'-bisphosphate to the 3'-hydroxyl group of a DNA oligomer. Using these reactions, we will synthesize three 24-base pair-long duplexes containing single nucleotide substitutions in the hexameric recognition site for Eco RI restriction endonuclease and methylase. In addition we will synthesize a series of compounds of the type Ado-5'PP-X in which X has variable ability to activate the beta-phosphate to nucleophilic attack. Since compounds of this general type are substrates for RNA ligase in an ATP-independent reaction, their use with the enzyme should clarif the electronic and stereochemical mechanisms of the enzyme. Other beta-ADP derivatives with fluorescent compounds and biotin will be synthesized and tested with the enzyme with the aim of developing an enzymatic method of modifying the 3'-termini of nucleic acids.